Anisotropic molecular rotational diffusion in 15N spin relaxation studies of protein mobility

The backbone dynamics of the uniformly 15N-labeled N-terminal 63-residue DNA-binding domain of the 434 repressor has been characterized by measurements of the individual 15N longitudinal relaxation times, T1, transverse relaxation times, T2, and heteronuclear 15N[1H]-NOEs at 1H resonance frequencies of 400 and 750 MHz. The dependence of an apparent spherical top correlation time, tauR, on the orientation of the N-H bond vector with respect to the principal axes of the global diffusion tensor of the protein was used to establish the fact that the degree of anisotropy of the global molecular tumbling amounts to 1.2, which is in good agreement with the values obtained from model calculations of the hydrodynamic properties. A model-free analysis showed that even this small anisotropy leads to the implication of artifactual slow internal motions for at least two residues when the assumption of isotropic global motion is used. Additional residues may actually undergo internal motions on the same time scale as the global rotational diffusion, in which case the model-free approach would, however, be inappropriate for quantifying the correlation times and order parameters. Overall, the experiments with 434(1-63) demonstrate that the assumption of isotropic rotational reorientation may result in artifacts of model-free interpretations of spin relaxation data even for proteins with small deviations from spherical shape.     

Solution NMR characterization of hydrogen bonds in a protein by indirect measurement of deuterium quadrupole couplings

Hydrogen bonds stabilize protein and nucleic acid structure, but little direct spectroscopic data have been available for characterizing these critical interactions in biological macromolecules. It is demonstrated that the electric field gradient at the nucleus of an amide hydrogen can be determined residue-specific by measurement of 15N NMR relaxation times in proteins dissolved in D2O, and uniformly enriched with 13C and 15N. In D2O, all backbone amide protons can be exchanged with solvent deuterons, and the T1 relaxation rate of a deuteron is dominated by its quadrupole coupling constant (QCC), which is directly proportional to the electric field gradient at the nucleus. 2HN T1 relaxation can be measured quantitatively through its effect on the T2 relaxation of its directly attached 15N. QCC values calculated from 2HN T1 and previously reported spectral densities correlate with the inverse cube of the X-ray crystal structure-derived hydrogen bond lengths: QCC = 228 + Sigmai 130 cos alphai/ri3 kHz, where alpha is the N-H...Oi angle and r is the backbone-backbone (N-)H...Oi(=C) hydrogen bond distance in angstroms.     

TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.     

Probing molecular motion by NMR

Recently developed solution NMR methods for measuring 2H, 13C, and 15N spin relaxation, coupled with biosynthetic isotopic enrichment, permit the characterization of backbone and sidechain dynamical properties of proteins on picosecond/nanosecond and microsecond/millisecond timescales. Theoretical interpretations of the relaxation data provide insights into the biophysical and functional properties of proteins.     

Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II

Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.     

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.     

Photoproducts of bacteriorhodopsin mutants: a molecular dynamics study

Molecular dynamics simulations of wild-type bacteriorhodopsin (bR) and of its D85N, D85T, D212N, and Y57F mutants have been carried out to investigate possible differences in the photoproducts of these proteins. For each mutant, a series of 50 molecular dynamics simulations of the photoisomerization and subsequent relaxation process were completed. The photoproducts can be classified into four distinct classes: 1) 13-cis retinal, with the retinal N-H+ bond oriented toward Asp-96; 2) 13-cis retinal, with the N-H+ oriented toward Asp-85 and hydrogen-bonded to a water molecule; 3) 13,14-di-cis retinal; 4) all-trans retinal. Simulations of wild-type bR and of its Y57F mutant resulted mainly in class 1 and class 2 products; simulations of D85N, D85T, and D212N mutants resulted almost entirely in class 1 products. The results support the suggestion that only class 2 products initiate a functional pump cycle. The formation of class 1 products for the D85N, D85T, and D212N mutants can explain the reversal of proton pumping under illumination by blue and yellow light.     

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.     

Self-association and backbone dynamics of the hck SH2 domain in the free and phosphopeptide-complexed forms

Decreased dynamic motion in the peptide backbone of proteins may accompany ligand binding and influence the thermodynamic and kinetic stability of the resulting complexes. We have investigated the diffusional behavior and backbone dynamics of the free and phosphopeptide (EPQpYEEIPIYL) complexed Hck SH2 domain using NMR spectroscopy. Both the free domain and its phosphopeptide complex self-associate at higher protein concentrations. Diffusional measurements and surface analysis indicate that charged side-chain groups are probably responsible for self-association. Higher order aggregation, such as trimer and tetramer, also occurs at elevated protein concentrations. Dynamic motion in the peptide backbone of Hck SH2 was determined from 15N relaxation data fit using extended model-free parameters. The rotational correlation time (taum) for uncomplexed Hck SH2 was 6.8 ns while taum for peptide-bound Hck SH2 was 7.6 ns. Generalized order parameters (S2) increased for most residues upon binding of the phosphopeptide, consistent with peptide binding restricting motion of the NH bond vectors on the picosecond time scale. These studies suggest that complexation increases internal order in Hck SH2 and that internal dynamic motions contribute to the activation of Src-family kinases in vivo.     

Phase labeling of C-H and C-C spin-system topologies: application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins

Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH 'straight-through' triple-resonance experiments that allow rapid and almost complete assignments of backbone H(alpha), 13C(alpha), 15N and H(N) resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly C(alpha) and H(alpha) resonances: (i) C-H phase discrimination by tuning of the refocusing period tau(a_f); (ii) C-C phase discrimination by tuning of the 13C constant-time evolution period 2T(c). For small proteins, C-C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C-H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing 13C-13C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.     

Solution structure, rotational diffusion anisotropy and local backbone dynamics of Rhodobacter capsulatus cytochrome c2

The solution structure, backbone dynamics and rotational diffusion of the Rhodobacter capsulatus cytochrome c2 have been determined using heteronuclear NMR spectroscopy. In all, 1204 NOE-derived distances were used in the structure calculation to give a final ensemble with 0.59(+/-0.08) A rms deviation for the backbone atoms (C, Calpha and N) with respect to the mean coordinates. There is no major difference between the solution structure and the previously solved X-ray crystal structure (1.07(+/-0.07) A rms difference for the backbone atoms), although certain significant local structural differences have been identified. This protein contains five helical regions and a histidine-heme binding domain, connected by a series of structured loops. The orientation of the helices provides an excellent sampling of angular space and thus allows a precise characterization of the anisotropic diffusion tensor. Analysis of the hydrodynamics of the protein has been performed by interpretation of the 15N relaxation data using isotropic, axially asymmetric and fully anisotropic diffusion tensors. The protein can be shown to exhibit significant anisotropic reorientation with a diffusion tensor with principal axes values of 1.405(+/-0.031)x10(7) s-1, 1.566(+/-0.051)x10(7) s-1 and 1.829(+/-0.054)x10(7) s-1. Hydrodynamic calculations performed on the solution structure predict values of 1.399x10(7) s-1, 1.500x10(7) s-1 and 1.863x10(7) s-1 when a solvent shell of 3.5 A is included in the calculation. The optimal orientation of the diffusion tensor has been incorporated into a hybrid Lipari-Szabo type local motion-anisotropic rotational diffusion model to characterize the local mobility in the molecule. The mobility parameters thus extracted show a quantitative improvement with respect to the model-free analysis assuming isotropic reorientation; helical regions exhibit similar dynamic properties and fewer residues require more complex models of internal motion. While the molecule is essentially rigid, a tripeptide loop region (residues 101 to 103) exhibits flexibility in the range of 20 to 30 ps, which appears to be correlated with the order in the NMR solution structure.     

Protein rotational relaxation as studied by solvent 1H and 2H magnetic relaxation

Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.     

Measurement of the degree of coupled isotopic enrichment of different positions in an antibiotic peptide by NMR

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent 1H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between 13C and nearby sites that are only fractionally labeled with 15N, and measuring the fraction of molecules in which 13C is coupled to 15N, at each of several sites. The 15N of [2-13C, 15N]-labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, 15N and 13C are incorporated into the N11 and C10 positions together as an 15N13C fragment approximately 8% of the time, whereas 15N is incorporated largely independently at the other positions.     

PMR-relaxation and steric computations give unequivocal nucleoside conformations

The configuration and the conformation of alpha and beta anomers of pyrazomycin, cytidine and pseudouridine in aqueous solution have been investigated by 1H-NMR at 250 MHz. T1 proton relaxation measurements are an excellent method to determine the conformation of the base around the glycosidic linkage. Frequently, steric hindrance considerations can help to decide which conformations are possible in nucleoside anomer pairs. The proton-proton coupling constants indicate that the N conformer is largely predominant in the alpha anomers while the S conformer is particularly abundant in beta-pyrazomycin. The steric hindrance is much larger for alpha than for beta-nucleosides and change of a C-C to a C-N glycosidic bond reduces considerably the rotational possibilities of the base. The relaxation data show that alpha-cytidine adopts the anti conformation with gamma = 200 degrees in good agreement with the crystal structure and with the sterical computations. In the other case, when the syn and anti conformations are sterically accessible, the orientation of the base may be completely different from one nucleoside to the other. It can be predicted neither from the crystal structure nor from comparisons with similar compounds. For alpha-pseudo-uridine the predominant orientation of the base (gamma = 120 degrees) is in the boundary of the syn-anti regions; for beta-cytidine the syn (gamma = 65 degrees) and anti (gamma = 215 degrees) conformations are equiprobable at room temperature while beta-pseudouridine shows the syn conformation with gamma = 40 degrees, the smallest angle observed until now. There is no correlation between the N/S and syn-anti ratios.     

13C and 15N-chemical shift anisotropy of ampicillin and penicillin-V studied by 2D-PASS and CP/MAS NMR

The principal values of the chemical shift tensors of all 13C and 15N sites in two antibiotics, ampicillin and penicillin-V, were determined by 2-dimensional phase adjusted spinning sideband (2D-PASS) and conventional CP/MAS experiments. The 13C and 15N chemical shift anisotropies (CSA), and their confidence limits, were evaluated using a Mathematica program. The CSA values suggest a revised assignment of the 2-methyl 13C sites in the case of ampicillin. We speculate on a relationship between the chemical shift principal values of many of the 13C and 15N sites and the beta-lactam ring conformation.     

Developing an effective toxicology/quality assurance partnership. Improving quality, compliance, and cooperation

A novel approach to the synthesis of the orally active estrogen 14 alpha,15 alpha-methylene estradiol (8, J 824) is described, starting with 3-methoxy-estra-1,3,5(10),8,14-pentaen-17 alpha-ol (5). The 14 alpha, 15 alpha-methylene bridge was sonochemically introduced by regioselective and stereoselective Simmons-Smith methylenation of the 14-double bond. Birch reduction of the 8-double bond provided the desired 8 beta-H, 9 alpha-H steroid, whereas ionic hydrogenation afforded the 8 beta-H, 9 beta-H isomer, together with an epimerization of the 17 alpha-hydroxy group. Oxidation of the Birch product yielded the corresponding 17-oxo steroid, which gave the title compound by diborane reduction. For radioimmunoassay development the 6-(O-carboxymethyl)-oximino derivative of 8 was prepared as hapten and the 2-hydroxy derivative of 8 was synthesized as a potential metabolite of 8, and 8 was tritium labeled as well.     

Iterative lineshape fitting of MAS NMR spectra: a tool to investigate homonuclear J coupling in isolated spin pairs

Possibilities and limitations of iterative lineshape fitting procedures of MAS NMR spectra of isolated homonuclear spin pairs, aiming at determination of magnitudes and orientations of the various interaction tensors, are explored. Requirements regarding experimental MAS NMR spectra as well as simulation and fitting procedures are discussed. Our examples chosen are the isolated 31P spin pairs in solid Na4P2O7. 10H2O, (1), and Cd(NO3)2. 2PPh3, (2). In both cases the two 31P chemical shielding tensors in the molecular unit are related by C2 symmetry, and determination of the orientations of these two tensors in the molecular frame is possible. In addition, aspects of homonuclear J coupling will be addressed. For 1, both magnitude and sign of 2Jiso(31P, 31P) (Jiso = -19.5 +/- 2.5 Hz) are obtained; for 2, (Jiso = +139 +/- 3 Hz) anisotropy of J with an orientation of the J-coupling tensor collinear, or nearly collinear, with the dipolar coupling tensor can be excluded, while absence or presence of anisotropy of J with any other relative orientation of the J-coupling tensor cannot be determined.     

The fructose transporter of Bacillus subtilis encoded by the lev operon: backbone assignment and secondary structure of the IIB(Lev) subunit

The fructose transporter of the Bacillus subtilis phosphotransferase system consists of two membrane associated (IIA and IIB) and two transmembrane (IIC and IID) subunits [Martin-Verstraete, I., Débarbouille, M., Klier, A. & Rapoport, G. (1990) J. Mol. Biol. 214, 657-671] . It mediates uptake by a mechanism which couples translocation to phosphorylation of the transported solute. The 18-kDa IIBLev subunit transfers phosphoryl groups from His9 of the IIA subunit to the sugar. The three-dimensional structure of IIBLev or similar proteins is not known. IIBLev was overexpressed in Escherichia coli and isotopically labelled with 13C/15N in H2O as well as in 70% D2O. 15N-edited NOESY, 13C-edited NOESY and 13C,15N triple-resonance experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on qualitative interpretation of NOE, scalar couplings, chemical shift values and amide exchange data, the secondary structure and topology of IIBLev was determined. IIBLev comprises six parallel beta-strands, one antiparallel beta-strand and 5 alpha-helices. The order of the major secondary-structure elements is (beta alpha)5beta (strand order 7651423). Assuming that the (beta alpha beta)-motives form right-handed turn structures, helices alphaA and alphaB are packed to one face and helices alphaC, alphaD and alphaE to the opposite face of the parallel beta-sheet. His15 which is transiently phosphorylated during catalysis is located in the loop beta1/alphaA of the topological switch point. The amino terminal (beta/alpha)4 part of IIBLev has the same topology as phosphoglyceromutase (PGM; PDB entry 3pgm). Both proteins catalyze phosphoryltransfer reactions which proceed through phosphohistidine intermediates and they show a similar distribution of invariant residues in the topologically equivalent positions of their active sites. The protein fold of IIBLev has no similarity to any of the known structures of other phosphoenolpyruvate-dependent-carbohydrate-phosphotransferase-system proteins.